1. Modular construction of DNA aptamers for human thrombin
Zavyalova Elena
Kopylov Alexey
Apto-Pharm Ltd:
Moscow State University
Russian Academy of Sciences
PharmEco Holding 1 1 1 1 1 1 1 1

2. Molecular Recognition Elements (MoRE)‏ made of Nucleic Acids
Aptamers –
Molecular Recognition Elements (MoRE)‏
made of Nucleic Acids
Aptamers are oligonucleotides that share some attributes of monoclonal antibodies due to complex 3D structure
“Chemical Antibody” for Theranostics
(Therapy&Diagnostics) 2 2 2 2 2 2

3. 030213-141013 PubMed: aptamer + review = 37 refs (370 Total)
Mode of Application: 
1: Yadav SK, Chandra P, Goyal RN, Shim YB. A review on determination of steroids in biological samples exploiting nanobio-electroanalytical methods. Anal Chim Acta Jan 31;762:14-24.
 2: Wang T, Ray J. Aptamer-based molecular imaging. Protein Cell Oct;3(10):
 3: Sundaram P, Kurniawan H, Byrne ME, Wower J. Therapeutic RNA aptamers in clinical trials. Eur J Pharm Sci Jan 23;48(1-2):
 4: Xing H, Wong NY, Xiang Y, Lu Y. DNA aptamer functionalized nanomaterials for intracellular analysis, cancer cell imaging and drug delivery. Curr Opin Chem Biol Aug;16(3-4):
 5: Pednekar PP, Jadhav KR, Kadam VJ. Aptamer-dendrimer bioconjugate: a nanotool for therapeutics, diagnosis, and imaging. Expert Opin Drug Deliv Oct;9(10):
 6. Mishra S, Kim S, Lee DK. Recent patents on nucleic acid-based antiviral therapeutics. Recent Pat Antiinfect Drug Discov Nov 1; 5(3):
Field of Application:
1: Binning JM, Leung DW, Amarasinghe GK. Aptamers in virology: recent advances and challenges. Front Microbiol. 2012;3:29.
 2: Hu M, Zhang K. The application of aptamers in cancer research: an up-to-date review. Future Oncol Mar;9(3):369-76
 3: Yang Y, Ren X, Schluesener HJ, Zhang Z. Aptamers: Selection, Modification and Application to Nervous System Diseases. Curr Med Chem (27):
 4: Haberland A, Wallukat G, Schimke I. Aptamer binding and neutralization of β1-adrenoceptor autoantibodies: basics and a vision of its future in cardiomyopathy treatment. Trends Cardiovasc Med Aug;21(6):
 5: Vavalle JP, Cohen MG. The REG1 anticoagulation system: a novel actively controlled factor IX inhibitor using RNA aptamer technology for treatment of acute coronary syndrome. Future Cardiol May;8(3): 3 3 3

4. The success story:
ANGIOGENESIS - new vessels are created from pre-existing vasculature. Increased rates of angiogenesis are associated with several disease states:
- cancer
- age-related macular degeneration (AMD)
- psoriasis
- rheumatoid arthritis
- diabetic retinopathy
Treatment options for AMD
have been limited with photodynamic therapy
Commercial VEGF inhibitors/drugs are:
- RNA APTAMER pegaptanib (Macugen, Eyetech Parm/Pfizer)
partial and full length ANTIBODIES ranibizumab
(Fab, Lucentis, $1,600), and bevacizumab (Avastin, $40), Genentech
- VEGF receptor trap - fusion protein aflibercept
- small interfering RNA-based therapies bevasiranib and AGN ,
sirolimus
- and tyrosine kinase inhibitors, including vatalanib, pazopanib
TG , TG , AG , and AL 39324
Therapies have met with great success in reducing
the vision loss associated with neovascular AMD
Retina Feb;33(2): Intravitreal pegaptanib sodium (macugen) for treatment of myopic choroidalneovascularization: a morphologic and functional study.
1994 –
(NeXstar
Pharma)
- 2004
(FDA) 4 4 4 4 4 4 4 4

5. Next – NOXXON? http://www.regadobio.com/ Sept. 17, 2013 /PRNewswire/
REGADO BIOSCIENCES, INC.
ENROLLS FIRST PATIENT
IN PHASE 3 TRIAL OF REG1"REGULATE-PCI"
TO STUDY REG1 IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION
BASKING RIDGE, N.J.,
Sept. 17, 2013 /PRNewswire/
TIDES, May 2013, Boston
Regado Biosciences, Inc. (Nasdaq: RGDO) - discovery and development of novel, first-in-class, actively controllable antithrombotic drug systems for acute and sub-acute cardiovascular indications, today announced the enrollment of the first patient in its REGULATE-PCI clinical trial.  REGULATE-PCI is Phase 3, PROBE design (Prospective, Randomized, Open-label, Blinded-Endpoint) superiority study comparing the effects of Regado's REG1 to bivalirudin in patients undergoing percutaneous coronary intervention (PCI) electively or for the treatment of unstable angina (UA) or non-ST elevated myocardial infarction (N- STEMI).  REGULATE-PCI, if successful, will serve as the basis for product registration applications throughout the world. Led by co-PIs, Drs. J. H. Alexander of Duke University Medical Center, A. M.Lincoff of The Cleveland Clinic and R. Mehran of Mount Sinai School of Medicine, REGULATE-PCI is expected to enroll 13,200 patients at approximately 500 investigational sites worldwide.  The primary endpoint of the trial is efficacy compared to bivalrudin based on a composite of death, nonfatal myocardial infarction (MI), nonfatal stroke and urgent target lesion revascularization through day three.  The principal secondary endpoint is safety compared to bivalrudin as measured by major bleeding events through day three.  The trial is powered to show superiority in efficacy and non-inferiority in safety against bivalirudin.  If successful, REGULATE-PCI will become the cornerstone of Regado's international new drug applications, expected to be filed in early  The first of three key interim analyses in the trial will occur after enrollment of the first 1,000 patients and is expected to occur during the second quarter of 2014.
Next – NOXXON?

6. MAKING APTAMERS
SELEX - Systematic Evolution of Ligands by EXponential enrichment SELEX –in vitro selection of RNA/DNA
of single stranded oligo combinatorial libraries
for molecules which bind a target.
Winners, not champs
The molecules are coined APTAMERS [aptus (lat) – to correspond, to fit)
APTAMERS are single-stranded oligos with 3D structure that binds to the target with high affinity and specificity, and possibly modulate target function.
Goal of SELEX – to fish out aptamers, and to make large amount of aptamers by chemical synthesis
L. Gold,1990 6 6 6 6 6 6 6 6 6 6 6 6

7. SELEX Chemical synthesis and selection of aptamers out of 4n sequences
(ie 1014‏ for pegaptanib)
Families of aptamers
with repertoire
of affinities
Winners, but not champs 7 7 7 7 7 7 7 7 7 7

8. Aptus: “to fit”
mer : “smallest unit of repeating structure”
Aptamers could be developed for different targets, both LMW and HMW:
Toxins
Proteins
Viruses
Pathogenic microorganisms
Cancer cells 8 8 8 8 8 8 8 8 8

9. Aptamer targets Thrombin 9 Aptamers under development Oncology
Mycobacterium tuberculosis‏
Acetylcholine
(nicotine)
Viruses
Aptamers
under
development
Allergy ‏
Photodynamic and Radiotherapy
Prions
Alzheimer's disease
LMW comps & drugs
Thrombin 9 9 9 9 9 9 9 9 9 9 9 9

10. APTAMERS vs/and ANTIBODIES KNOWING APTAMERS
High affinity and specificity for the target
High affinity and specificity for the target
In vivo biological protocols
In vitro chemical protocols
Binding parameters could be modified
Possibility for changes of binding parameters
Reversible temperature denaturing
Irreversible temperature denaturing
Extended storage time
Limited storage time
Low immunogenity
High immunogenity
Availability of specific ANTIDOTE
NO rational ANTIDOTE
Aptamers have some potential advantages
Then why the progress slow? 10 10 10 10 10 10 10 10 10 10

11. Several Aptamers are on Clinical Trials Then why the progress slow?
A key attribute of THERAPEUTIC APTAMERS is the ability to tailor the pharmacokinetic profile by modulating the degree of metabolic stability, renal clearance and rate of distribution
Good safety margins between the pharmacologically effective dose and toxicologically established no-adverse-effect levels
Several Aptamers are on Clinical Trials
Then why the progress slow?
<aptamer>: 27/21 entries (oct, 2013/feb, 2012)
Why the progress is slow? 11 11 11 11 11 11 11

12. 2 Dual Paradigm of Drug Design
I. Small – CHEMICALS, Low molecular weight
Creation of combinatory library of synthetic and natural CHEMICAL COMPOUNDS
Selection by activity
Chemical synthesis
PLUS – Better distribution
MINUS – Less specificity
II. Large – BIOLOGICS, High molecular weight
Identification of proteins with defensive functions (Ig, IFN, GF)
Making genetic engineering constructs Biotechnological synthesis.
MINUS – Slow distribution
PLUS – High specificity 12 12 12 12 12 12 12 12 12 12 12

13. Triple Paradigm of Anti-Thrombin Drug Design
I. CHEMICALS
Dabigatran
(Pradaxa)
APTAMERS as the third paradigm
Intermediate size status in attempt
to combine the best of both groups
Peptamer: Hirulog (20 aa) (Bivalirudin, Angiomax, Angiox)‏
II. BIOLOGICS
Leeches > hirudin (65 aa) 13 13 13 13 13 13 13 13 13 13 13

14. Prophylactics and Treatments of Thrombosis
1. Thrombolytics – dissolve thrombi
(Streptokinase (SK), Urokinase (UK),
Tissue plasminogen activator (tPA)
2. Anti-aggregants – inhibits platelet aggregation
(aspirin, Thienopyridines – Clopidogrel (Plavix),
IIb-IIIa glycoproteins antagonists
3. ANTI-COAGULANTS – inhibit fibrin formation:
Non-direct anticoagulants (warfarin)
Direct anti-coagulants:
Heparin and derivatives (thrombin, 10a)
Enoxaparin (10a)
Rivaroxaban (10a)
Direct thrombin inhibitors:
monovalent – chemicals (dabigatran)
bivalent - biologics (bivalirudin) 14 14 14 14 14 14 14 14

15. anti-thrombin aptamer applications
Primary challenges of
anti-thrombin aptamer applications
to reduce
cerebral embolization
after
carotid endarterectomy
to reduce
post-operational bleeding
after
coronary artery bypass surgery 15 15 15 15 15 15 15 15 15 15 15

16. Coagulation Cascade and Aptamers
Willebrand Factor
RA36
REG2 16 16 16 16 16 16 16 16

17. Unique opportunity – fast antidot
Aptamer blocks thrombin, and antidot blocks aptamer, restoring coagulation
Fibrin mesh
for clotting 17 17 17 17 17 17

18. 4 steps of making useful aptamer
I. Selecting primary aptamer families
Gold Rush (Missing links):
Understanding aptamers
Designing aptamers
II. Adjusting aptamers to the target
III. Making aptamers stable/durable
IV. Solving specific tasks
V. Making therapeutic aptamer 18 18 18 18 18 18 18 18

19. G-quadruplex structure of 15-mer (15TGT) - chair
minimal DNA aptamer, pharmacophore
Cation (+) in the center 19 19 19 19 19 19 19 19

20. Which loop (TT or TGT) is a pharmacophore?
X-Ray of the complex of thrombin with 15TGT
Problem # 2
Which loop (TT or TGT) is a pharmacophore? 20 20 20 20 20 20 20 20

21. DNA aptamer and Thrombin
hydrolyzes
fibrinogen
which yields
clot formation
2) Aptamer inhibits
fibrinogen binding
and clot formation
Question # 3: Is it a competitive binding/inhibiting? 21 21 21 21 21 21 21 21

22. ? ? Understanding Aptamers Lack of Structure -Function Relationship
31-mer TGT‏ (Japan)
Ki 0,3 nM (RF)
26-mer NU 172 (USA)
15-mer ТGT‏ (USA)
Ki 14,7 nM (RF)
3D: X-ray, NMR
No 3D: just 1D
complex ?
MD: rational drug design
Apto-Pharm Ltd
RA-36‏ (RF)
Ki 7,5 nM (RF) 22 22 22 22 22 22 22 22 22

23. DNA aptamer modelling with molecular dynamics using super-computer
‘Lomonosov’ Top 31 (06/2013)
Cores: 78,660; Rmax (901.9 TFlop/s)
NO conventional force field parameters were available till now
Porting of new Force Fields into Gromacs 23 23 23 23 23 23 23 23 23

24. T12 and T3 interact with the thrombin
Bottom view of
2 TT base pairs of
2 TT loops
NMR model of 15TGT during MD
in the new force field
T12 and T3 interact with the thrombin 24 24 24 24 24 24 24 24 24

25. TGT upper loop vs TT bottom loops Loops vs cation MD: 60-80 ns frame
Na+ movement into
G-quadrulpex
through TT loops
Most of the time
Na+ sits in the center
of G-quadruplex

26. Aptamer – cation QM/MM simulation
ps scale
Aptamer – cation QM/MM simulation
QM/MM simulation approach for proteins:
Martin Karplus, Michal Levitt, Arieh Warshell,
Nobel Prize in Chemistry 2013, Oct 9

27. Functional Aptamers: Io & Coagul Activity
Just putative structures
of 15TGT pharmacophore extentions
31TGT
NU172
no IIIo
single IIIo
RA36
15TGT

28. MD for exploring and design of extended pharmacophore structures
[G-quadruplex + Duplex?] А B 28 28 28 28 28 28 28 28

29. Structure, 3D Assembly 29 Duplex is stable enough G-quadruplex has
UV melting
of 15TGT
and 31TGT
(260 nm)‏
G-quadruplex has
the same
thermal stability
within 15TGT and 31TGT
CD melting of 15TGT and 31TGT (294 nm)‏ 29 29 29 29 29 29 29 29 29

30. Affinity. Kinetics of Interactions:
thrombin + 15-mer or 31-mer
Surface Plasmon Resonance
Kon different, Kof similar

31. Function:
Kinetics of Fibrinogen Hydrolysis
with Thrombin

32. Thrombin hydrolyzes fibrinogen, and fibrin aggregates
Thrombin hydrolyzes fibrinogen, and fibrin aggregates. Models, AFM, optical density measurements 32 32 32

33. Inhibition constant, nM
Possibility of
calculations of both Ki and Inhibition Type
Examples:
bivalirudin
hirudin
heparin
aptamer
Aptamer
Inhibition type
Inhibition constant, nM
15ТGT
Non-competitive
14,7±1,0
31ТGT
Competitive
0,34±0,10
NU172
0,29±0,06
RA-36
7,5±0,3 33 33 33

34. Apta-nano-Lego a J j
31TGT
15TGT j j a 34 34 34

35. Apta-nano-Lego
15TGT
31TGT
NU172
Ki, nM 14,7 0, , , ,

36. Ki, nM 7,5 Making multi-nano-tools Bivalent Aptamer , RA-36 Apto-Pharm
The inhibiting type
not like for extended aptamers
PCT WO 2011/ A1, Dec 13, 2010 36 36 36

37. Blood plasma tests for RA-36: species specificity
INR = 3 (like for coumarin) 37 37 37 37 37 37 37 37 37

38. Thrombin Generation Assay
control
[thrombin], nM
Time, s

39. Blood Clot Mouse model for venous thrombosis
RA-36 inhibits clot formation: vessel cross-sections
Blood
Clot

40. Short duration time – several min
Animal tests:
Short duration time – several min
Thrombin time, s 40 40 40 40 40 40 40 40 40

41. PCT WO 2011/075004 A1, Dec 13, 2010 Affinity Modelling Synthesis
CURRENT STATUS: formal pre-clinical trials
1st Moscow State Medical University 1 3
Modelling
Affinity
Synthesis
Animal trials Coagulation Inhibition
PCT WO 2011/ A1, Dec 13, 2010 41 41 41 41 41 41 41 41 41 41 41 41

42. Apto-Pharm Ltd: www.apto-parm.com
Kopylov A
(V Dir Sci)‏
Golovin A (Chem Dpt)‏
Reshetnikov R
Savelyeva S
Turashev A
Turchaninov T
Yuminova A
Zavyalova E
Pavlova G (Biol Dpt)‏
Khairulina G
Kust N
Pustogarov N
Revischin A
Suvchenko E
Susova O
Samoylenkova N
Special thanks for Thomas Rupp, Consultant 42 42 42

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